SPA-Cys
Staphylococcal Protein-A Cys Recombinant
Cat. No.
BT10327
Source
Escherichia Coli.
Synonyms
Immunoglobulin G-binding protein A, IgG-binding protein A, Staphylococcal protein A, SPA.
Appearance
Sterile Filtered White lyophilized (freeze-dried) powder.
Purity
Greater than 98.0% as determined by:
(a) Analysis by HPLC.
(b) Analysis by SDS-PAGE.
Usage
Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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Description
SPA-Cys Recombinant produced in E.Coli is a single non-glycosylated polypeptide chain with a Cys on C-terminus. SPA-Cys is comprised of 5 IgG-binding domains E-D-A-B-C aligned in series containing 297 amino acids and having a molecular mass of 33.5kDa containing little or no carbohydrate. Cell wall binding region, cell membrane binding region and albumin binding region were removed to ensure the highest specific IgG binding.
Product Specs
Introduction
Protein A is a vital component found in the cell walls of certain Staphylococcus aureus strains. It plays a crucial role in binding to antibodies, specifically the IgG class. The recombinant version of Protein A, engineered for research purposes, retains the five IgG-binding regions of the native protein, making it highly effective for purifying both polyclonal and monoclonal IgG antibodies. This recombinant Protein A exhibits similar functionality to its native counterpart, effectively binding to specific IgG subclasses from various species, including human IgG1, IgG2, and IgG4; mouse IgG2a, IgG2b, and IgG3; and rat IgG2c. Furthermore, it demonstrates binding affinity for total IgG from rabbit, pig, dog, cat, and guinea pig.
Description
SPA-Cys Recombinant, produced in E.Coli, is a single, non-glycosylated polypeptide chain with a C-terminal Cys residue. This protein consists of five tandem IgG-binding domains (E-D-A-B-C), encompassing 297 amino acids, and has a molecular weight of 33.5 kDa. Notably, it contains minimal to no carbohydrates. To optimize specific IgG binding, regions associated with cell wall binding, cell membrane binding, and albumin binding have been removed.
Physical Appearance
Sterile Filtered White lyophilized powder.
Formulation
The SPA protein was lyophilized without any additional additives.
Solubility
For reconstitution of lyophilized SPA, sterile 18MΩ-cm H2O is recommended, with a minimum concentration of 0.1mg/ml. Further dilutions can be prepared in other aqueous solutions as needed.
Stability
Lyophilized SPA, while stable at room temperature for up to 3 weeks, should ideally be stored in a desiccated state below -18°C. After reconstitution, it can be stored at 4°C for 2-7 days. For long-term storage, freezing below -18°C is recommended, preferably with the addition of a carrier protein like HSA or BSA (0.1%). Avoid repeated freeze-thaw cycles.
Purity
The purity of SPA-Cys is greater than 98.0%, as determined by High Performance Liquid Chromatography (HPLC) and SDS-PAGE analysis.
Synonyms
Immunoglobulin G-binding protein A, IgG-binding protein A, Staphylococcal protein A, SPA.
Source
Escherichia Coli.
Amino Acid Sequence
NAAQHDEAQQ NAFYQVLNMP NLNADQRNGF IQSLKDDPSQ SANVLGEAQK LNDSQAPKAD AQQNNFNKDQ QSAFYEILNM PNLNEAQRNG FIQSLKDDPS QSTNVLGEAK KLNESQAPKA DNNFNKEQQN AFYEILNMPN LNEEQRNGFI QSLKDDPSQS ANLLSEAKKL NESQAPKADN KFNKEQQNAF YEILHLPNLN EEQRNGFIQS LKDDPSQSAN LLAEAKKLND AQAPKADNKF NKEQQNAFYE ILHLPNLTEE QRNGFIQSLK DDPSVSKEIL AEAKKLNDAQ APKEEDC
Product Science Overview
Structure and Function
The primary structure of SPA consists of five homologous domains, each capable of binding to the Fc region of IgG. This binding is highly specific and occurs through a non-covalent interaction, which is crucial for various applications, including antibody purification and immunoprecipitation.
The introduction of an engineered cysteine residue in SPA allows for site-specific conjugation. This modification is particularly useful for immobilizing the protein on solid supports, such as in biosensors or chromatography resins. The cysteine residue provides a reactive thiol group, which can form covalent bonds with other molecules, enhancing the stability and functionality of the conjugated protein.
Production and Challenges
SPA-Cys can be produced through intracellular or extracellular expression in Escherichia coli (E. coli). However, the production process presents several challenges:
- Dimerization: During production, the engineered cysteine residues can undergo spontaneous oxidation, leading to the formation of dimers. These dimers are often inactive and need to be reduced to monomers before further use .
- Monomeric Content: The efficiency of SPA-Cys in applications depends on the monomeric content. Intracellular production methods have been shown to yield higher monomeric content (≥85%) compared to extracellular methods, which often result in less than 50% monomeric content .
Applications
SPA-Cys has a wide range of applications in biotechnology and medical research:
- Antibody Purification: SPA-Cys is commonly used in affinity chromatography to purify antibodies from complex mixtures. The high specificity of SPA for the Fc region of IgG ensures efficient and selective purification.
- Biosensors: SPA-Cys can be immobilized on sensor surfaces to create biosensors for detecting immunoglobulins. These biosensors are highly sensitive and selective, making them valuable tools for diagnostic applications .
- Immunoprecipitation: SPA-Cys is used in immunoprecipitation assays to isolate and study specific proteins or protein complexes from cell lysates. The ability to conjugate SPA-Cys to solid supports enhances the efficiency of these assays.
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